Influenza virus belongs to the family Orthomyxoviridae family and is a negative strand, single-stranded RNA virus. The size of the viral particle is 80 to 120 nm. The viral particle has erythrocyte agglutinin (hemagglutinin, HA), neuraminidase (NA) and matrix protein (M2) in the lipid bilayer membrane of surface layer, which is lined with matrix protein 1 (M1). A segmented negative strand RNA within the surface layer forms a complex (RNP) with a nuclear protein (NP) and an RNA polymerase (PA, PB1, PB2). Influenza virus is classified into type A, B or C depending on antigenicity of the internal protein, among which type A and type B may cause an epidemic of influenza in human. It is type A that may cause a pandemic of influenza with potent pathogenicity. For type A influenza virus, it is known that there are many serotypes resulting from combination of 16 subtypes for HA and 9 subtypes for NA.
It is known that, in general, an RNA virus is susceptible to mutation. Influenza virus is not an exception and its antigenicity has been changed by degrees year by year through point mutation of a gene encoding HA or NA (antigen drift). For influenza virus type A, it is known that an antigenically different new virus strain develops by discrete mutation replacing either or both of HA and NA with another subtype(s) at an interval of several decades (antigen shift). Influenza viral mutation by such antigen drift and/or shift continually causes damages to human. In the past, several shifts of antigenicity had occurred in the world, to cause epidemic (pandemic) producing many victims. Specifically, there were Spanish flu in 1918, Asian flu in 1957, Hong-Kong flu in 1968 and Russian flu in 1977.
Influenza caused by infection of influenza virus is one of serious infectious diseases which occur in epidemics on a worldwide scale. There have been many cases of death or encephalitis in the aged, children or patients with a weak immune system. Even if influenza virus infection does not result in death in patients, physical symptoms such as fever, headache and fatigue, may force patients to stop social activities for a certain period of time resulting in great economic loss. Thus, there is a need for establishing an effective preventive measure against even a new virus strain, because of increased likelihood of occurrence of a new virus strain by shift of antigenicity in addition to importance of conventional protection against influenza virus infection.
A preventive measure against influenza is to perform vaccination every year. Vaccines currently in practical usage are split vaccines which comprise as a main ingredient HA purified from a strain for preparation of a vaccine cultured with chicken embryonated eggs. Thus, a virus predicted to occur in epidemics in the year is used as a strain for preparation of a vaccine. It is therefore necessary to determine a strain for preparation of a vaccine every year by predicting a strain causing epidemics, and in case of mismatch between a strain for preparation of a vaccine and a strain causing epidemics, the vaccine would be less effective. As compared to vaccines giving immunity over at least several years with a single vaccination schedule such as DPT (diphtheria, pertussis, tetanus) vaccine and Japanese encephalitis vaccine, current influenza vaccines need vaccination every year and therefore are inconvenient for both those who receive the vaccine and physicians who inject the vaccine and would be a burden of the expense. Furthermore, in case of occurrence of a different virus strain from an expected virus strain due to failure in the prediction in epidemics, those who receive the vaccine would still be infected with influenza virus in spite of vaccination. In particular, in case of occurrence of a new strain of influenza generated by shift resulting in significantly different antigenicity, since protective effects would scarcely be expected with the conventional influenza vaccines, explosive prevalence of the virus would result, so-called pandemic.
As described above, since current influenza vaccines can not sufficiently cope with mutation in the virus, a universal influenza vaccine less affected by antigenic mutation is highly desired to resolve such problem.
Since influenza virus causing such epidemic is influenza virus type A, a desired influenza vaccine against not only annual epidemic but also pandemic would be obtained by developing a vaccine that provides immunogenicity common in influenza virus type A. From such a point of view, research of a vaccine which targets a M2 protein common in influenza virus type A has been performed (See, for example, Non-patent reference 1). The M2 protein is a viral surface protein with a relatively well-conserved amino acid sequence among influenza virus type A. It is present in a relatively small amount in influenza virus particles (See, for example, Non-patent reference 2) but is expressed at a relatively high level in virus-infected cells (See, for example, Non-patent reference 3).
It has been reported that an antibody against M2 inhibits the replication of influenza virus type A both in in vivo and in vitro models (See, for example, Non-patent references 4 and 5). Further, Slepushkin et al., have reported that, in mice inoculated with M2, fatal infection by heterologous influenza virus type A is prevented and removal of the virus from the lung tissue is facilitated (See, for example, Non-patent reference 1). It has also been reported that a modified M2 protein in which a hydrophobic transmembrane domain is eliminated is useful for preparation of a vaccine (See, for example, Patent reference 1).
On the other hand, Neirynck et al., have reported that an extracellular domain of M2 fused to the N-terminal of a hepatitis B virus core antigen is used as a vaccine antigen (See, Non-patent reference 6). According to Neirynck et al., a hepatitis B virus core particle exposing M2 onto its surface is expressed in E. coli, the particle is purified from the E. coli, and an antibody against M2 is induced by administering the particle together with adjuvant.
Also, with an experiment using mice, Wu et al. (See, for example, Non-patent reference 7) and Mozdzanowska et al. (See, for example, Non-patent reference 8) have reported that a peptide (M2e) which corresponds to a region consisting of 23 amino acid residues generated after removal of a hydrophobic transmembrane domain from M2 also be able to protect the fatal infection by heterologous influenza virus type A by utilizing adjuvant or a peptide of oligomer called MAP. It is also revealed that at least one of epitopes against protective antibody produced by immunization with M2e is present in an amino acid region of from positions No. 6 to No. 13 of M2e (See, for example, Non-patent reference 9).
Preparation of the conventional influenza vaccines required time-consuming and laborious processes, i.e. first predicting a strain of virus prevalent in the year, adapting the virus to culture in eggs, culturing the virus in a large number of eggs, isolating the virus from the culture, inactivating the virus, and purifying an antigen protein. Development of a new vaccine that makes it unnecessary to predict a virus strain prevalent in the year so as to prepare a vaccine strain and that may cope with pandemic would greatly contribute to national welfare and reduce medical expense.    Patent reference 1: U.S. Pat. No. 6,169,175    Patent reference 2: JP-A-2001-512748    Non-patent reference 1: Slepushkin et al., 1995, Vaccine 13: p 1399-1402    Non-patent reference 2: Zebedee and Lamb, 1988 J. Virol. 62: p 2762-2772    Non-patent reference 3: Lamb et al., 1985 Cell 40: p 627-633    Non-patent reference 4: Hughey et al., 1995 Virology 212: p 411-421    Non-patent reference 5: Treanor et al., 1990 J. Virol. 64: p 1375-1377    Non-patent reference 6: 1999 Nature Med. 5: p 1157-1163    Non-patent reference 7: 2007 Vaccine 25: p 8868-8873    Non-patent reference 8: 2007 Virology J. 4: 118 doi: 10.1186/1743-422X-4-118    Non-patent reference 9: Wanli et al., 2004 Immunol. Lett 93: p 131-136